Download Discs large 5, an Essential Gene in Drosophila, Regulates Egg

INVESTIGATION
Discs large 5, an Essential Gene in Drosophila,
Regulates Egg Chamber Organization
Eve Reilly,* Neha Changela,* Tatyana Naryshkina,* Girish Deshpande,† and Ruth Steward*,1
*Department of Molecular Biology, Rutgers University, Waksman Institute, Cancer Institute of New Jersey, Piscataway,
New Jersey 08854 and †Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
ABSTRACT Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as
molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has
been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive
vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. We have
identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of
primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss
of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages.
Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes,
including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion,
E-cadherin, shown to be essential for proper organization of egg chambers.
Discs large 5 (Dlg5) is a member of the membrane-associated guanylate
kinase (MAGUK) family of proteins that typically serve as molecular
scaffolds and mediate signaling complex formation and localization.
MAGUK family proteins are highly conserved and carry a core PDZSH3-GUK domain. They are divided into 10 subfamilies, including the
discs large subfamily, named for homology to Drosophila discs large
(Lewander et al. 1990). PDZ (PSD-95, Discs Large, ZO 1) domains
are composed of approximately 100 amino acid residues and are often
localized to the plasma membrane (Oliva et al. 2012). Many PDZ domains are involved in assembly of signaling complexes in signal transduction pathways and cell polarity determination, and they have been
implicated in tumorigenesis (Oliva et al. 2012; Subbaiah et al. 2011).
First identified in humans, dlg5 was originally named P-dlg due to its
expression in the placenta and the prostate gland and for its homology
to Drosophila discs large (dlg1). Dlg1 is the founding member of the
Discs Large family, first studied because of its overgrowth phenotype;
Copyright © 2015 Reilly et al.
doi: 10.1534/g3.115.017558
Manuscript received February 20, 2015; accepted for publication March 15, 2015;
published Early Online March 19, 2015.
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
suppl/doi:10.1534/g3.115.017558/-/DC1
1
Corresponding author: Waksman Institute, 190 Frelinghuysen Road, Piscataway,
NJ 08854. E-mail: [email protected]
KEYWORDS
Drosophila
oogenesis
stalk and polar
cells
embryonic germ
cells
E-cadherin
mutant larvae fail to metamorphose and die with highly overgrown
discs (Woods and Bryant 1989). Dlg1 is a key regulator of apico-basal
polarity in neuroblasts and other polarized processes such as cell invasion (Roberts et al. 2012). Similar to dlg1, dlg5 has been demonstrated
to function during determination and maintenance of epithelial cell
polarity, during cell adhesion, migration, and proliferation, and during
signaling in vertebrates (Liu et al. 2014).
Polymorphisms in Dlg5 interfere with its scaffolding function and
are associated with inflammatory bowel disease in humans (Stoll et al.
2004). Despite this association, little is known about Dlg5. Drosophila
Dlg5 and human Dlg5 (Discs large 5 homolog) share the same general
domain architecture, with several conserved protein-binding domains.
Alignment of the amino acid sequences of homologous regions of
Drosophila and human Dlg5 shows that they share 45–54% sequence
homology (Roberts et al. 2012). Thus, dlg5 may be functionally conserved in flies and mammals, as is characteristic of many of the other
MAGUK proteins.
In vertebrates, Dlg5 is responsible for polarization of neural
progenitors and associates with Rab11-positive vesicles in epithelial
cells to serve as a scaffold for polarized delivery of cadherin-catenin
containing complexes to the plasma membrane (Nechiporuk et al.
2007) (Chang et al. 2010). In Drosophila, Dlg5 maintains cluster cohesion among migrating border cells in the ovary (Aranjuez et al.
2012). How Dlg5 exerts this function, however, remains largely unknown. Given the importance of the polarizing influence of Dlg5 in
other species (Liu et al. 2014), we decided to further investigate the role
of dlg5 in Drosophila oogenesis.
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Oogenesis in Drosophila begins when a germline stem cell (GSC),
located at the tip of the germarium (Figure 1A), undergoes an asymmetric division, forming a cystoblast and a GSC. After four divisions
with incomplete cytokinesis, the cystoblast ultimately forms a 16-cell
cyst. While moving through the germarium, the dividing cystoblast is
surrounded by somatic escort cells. At the transition from germarium
region 2a to 2b, these escort cells are exchanged with a monolayer of
follicle cells, resulting in the formation of a discrete egg chamber
(Figure 1A) (Morrison and Spradling 2008). Egg chamber formation
requires three crucial steps: first, follicle cell proliferation as a result of
asymmetric division of follicle stem cells (FSCs); next, follicle cell migration and encapsulation of the cyst; and, finally, differentiation of
stalk and polar cells to separate the newly formed egg chamber from its
antecedent (Dobens and Raftery 2000). Stalk cells and polar cells cease
dividing in the germarium soon after cell-fate specification, unlike
follicle cells that proliferate until approximately stage 7 (Tworoger
et al. 1999; Margolis and Spradling 1995; Zhang and Kalderon
2000). Egg chambers continuously "bud off" from the germarium
and move posteriorly as they continue to develop.
Here, we show that dlg5 is essential in embryos, and by clonal
analysis we find that dlg5 is required in FSCs and GSCs, because
persistent dlg5 germline and follicle cell clones were not detected.
Knockdown (KD) of dlg5 in FSCs and their daughters resulted in
egg chambers that never developed beyond stage 6. Phenotypes include ectopic polar cell clusters, stalk cell overgrowth, and the formation of disorganized egg chambers, sometimes containing two 16-cell
cysts. We further show that the distribution of E-cadherin in Dlg5 KD
follicle cells is affected. The phenotype associated with depletion of
Dlg5 in somatic cells of the ovary ultimately agrees with the phenotype
observed in dlg5 null clones. In both genetic backgrounds, mutant
clones and cells depleted of Dlg5 are eventually lost. Finally, dlg5
embryos display germ cell loss specifically during mid to late stages
Figure 1 Dlg5 in ovaries. (A) Drawing
of Drosophila ovariole that contains
a germarium and three egg chambers.
GSC, germline stem cells; FSC, follicle
stem cells; CC, cap cells; EC, escort
cells; Sp, spectrosome; TF, terminal filament. (B, B9) Ovariole expressing
Dlg5::GFP under the control of the
tub-Gal4 driver. Note the punctate
apical localization of Dlg5 in follicle
cells. FasIII marks follicle cells. (C) Control ovariole, driver only. (D) Ovariole
with reduced levels of Dlg5 in follicle
cells (Dlg5 KD). Note the long, multicellular stalks (arrowhead) and degenerate egg chambers (arrow). Scale bar,
20 mm.
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of embryogenesis, suggesting a requirement of dlg5 during germ cell
migration and gonad coalescence.
MATERIALS AND METHODS
Fly strains and generation of a dlg5 null allele
The fly stock expressing GFP-tagged Dlg5 (w; P{w[+mC]=UASCG6509.GFP}3), deposited by H. Richardson, was obtained from the
Bloomington Stock Center, as was the dlg5 RNAi line primarily used in
these experiments (UAS-CG6509GD11943RNAi) and the CyO balancer
expressing beta-galactosidase under the engrailed (en) promoter, CyO
en-LacZ (CyO, P{en1}wgen11). Additional dlg5 RNAi lines (P{GD11943}
v22496 and P{KK104086}VIE-260B) were obtained from VDRC. The
KD efficiency of P{GD11943}v22496 was assessed previously (Aranjuez
et al. 2012). Because KD of UAS-CG6509GD11943 and P{GD11943}
v22496 resulted in comparable phenotypes, we concluded that both
lines affect dlg5 expression to similar degrees. The dlg5D48 allele was
generated by imprecise excision of a P{SUPor-P}CG6509KG00748
(dlg5KG00748, Bloomington Stock Center) transposable element. To excise
the P-element, the insertion stock was crossed with a stock expressing
transposase (McGregor et al. 2002; Robertson et al. 1988). Flies carrying
potential excision chromosomes were crossed to w118; Df(2L)BSC242/
CyO, a deficiency stock uncovering the dlg5 gene region (Bloomington
Stock Center). Putative dlg5 mutants were identified by lethality.
To determine the lethal period of the excision alleles ,they were
crossed to a CyO balancer carrying a transgene that expresses GFP under
the ubiquitous (ubi) promoter (CyO, P{Ubi-GFP.S65T}PAD1, obtained
from the Bloomington stock center). These flies were crossed with w118;
Df(2L)BSC242/ CyO, P{Ubi-GFP.S65T}PAD1 and set-up for egg-laying
on agarose plates. The plates were changed after 8 hr and inspected for
survival of nongreen first instar larvae after 24 hr. At least 200 green
balancer larvae were counted for each cross.
The c587-Gal4;UAS-CD8::GFP driver was obtained from T. Schüpbach,
Princeton University; when GFP expression is not shown in the Figures,
we refer to this driver as c587-Gal4. All other drivers were obtained from
the Bloomington Stock Center.
Clonal analysis
Wild-type negatively marked clones were induced and analyzed as
described byFox et al. (2008). Newly eclosed adult y w P[hs70FLP];
ubi-GFP.nls FRT 40A/FRT 40A dlg5D48 flies were exposed twice within
24 hr to 38 for 1 hr per exposure. Mutant clones were induced in y w
P[hs70FLP];ubi-GFP.nls FRT 40A/FRT 40A dlg5D48 flies. Persistent
clones were induced and inspected at least three times, but the clones
were counted in only one experiment. Ubi-GFP.nls FRT40A flies were
obtained from the Bloomington Stock Center and y w P[hs70FLP] flies
were obtained from K. McKim, Rutgers University.
Immunostaining
Dissection, fixing, and staining of Drosophila ovaries were performed
as described previously (Minakhina et al. 2014). Primary antibodies
and concentration used, respectively, are as follows: guinea pig antiTraffic Jam (1:5000; a gift from D. Godt, University of Toronto),
mouse anti-FasIII [1:20; Developmental Studies Hybridoma Bank
(DSHB)], rat anti-Vasa (1:20), rat anti-DEcad (E-cadherin, 1:150;
DSHB), mouse anti-Eyes Absent (1:100; DSHB), and mouse anti3A9 (alpha spectrin, 1:20; DSHB). Phalloidin 488 (Invitrogen) and
secondary antibodies (Jackson Laboratories) were used at concentrations of 1:300. Hoechst 33258 (1:5000) was used to stain DNA. Images
were captured using a Leica DM IRBE SP2 or Leica TSC SP5 laser
scanning confocal microscope (40· oil objective), analyzed with Leica
Microsystems Software, and further processed using Adobe
Photoshop.
To observe germ cell migration, 0- to 16-hr-old embryos from the
dlg5/CyO engrailed-lacz stock were collected and fixed using 4%
Paraformaldehyde/Heptane mix. Embryos were stained with rabbit
anti-Vasa (1:1000; gift from Paul Lasko, McGill University) and rabbit
anti-B-galactosidase antibodies (purchased from Cappel; 1:10,000).
Subsequently, horseradish peroxidase–coupled anti-Rabbit secondary
antibody was used and the signal was visualized using standard histochemical procedures. Vasa antibody labels the primordial germ cells.
The homozygous mutant embryos were identified by the absence of
beta-galactosidase signal (Deshpande et al. 2009).
Mapping the dlg5D48 deletion
PCR analysis was used to determine the breakpoints of the deletion
generated in the P-element excision mutant dlg5D48. Because of the
perdurance of maternally contributed GFP, DNA from single dechorionated embryos was used for this analysis. PCR of DNA isolated from
wild-type and mutant embryos was done in parallel (Mathew et al.
2009). The P-element excision mutant stock was maintained over
a GFP expressing CyO balancer (CyO, P{Ubi-GFP.S65T}PAD1); homozygous mutant embryos were identified by selecting for lack of GFP at
4-6 hr after egg-lay, as soon as GFP-negative embryos could be identified.
Primer sequences:
Primer 6: 59-AAA AGG TTG TTA GTG CGT GCA-39
Primer 11: 59- GTG TGA CCG CGG AGT GAC-39
Primer 5: 59- TCACACTGGTGACGTTTTA-39
Primer 4: 59- CGCTCCCGGTAGTAGTTG-39
Primer 10: 59-CTG CAG GCG CAG TAC AAG TC-39
Primer 3: 59-TGA TGG TGC GGA TGG TAG-39
Primer 27: 59- AGT ATG TGG CAT TAA CAT GCG GG-39
Primer 28: 59- GAG TCG CCA GGC TCG TCT TTA-39
Primer 32: 59-GAC TTC TCC AAC CTC TTC-39
Primer 1: 59- GTT GCT TAG CTA GTC GCG-39
Primer 23: 59- TCC AGA ACG CCT ATG CTT TCG-39
Primer 24: 59- CGG AAG AGT GGT ATC GCC CA-39
RESULTS
Dlg5 is localized in follicle cells
Dlg proteins have been shown to localize asymmetrically within
tissues (Woods et al. 1996). To investigate Dlg5 protein distribution in
Drosophila ovarian tissue in the absence of a specific anti-Dlg5 antibody, we expressed GFP-tagged Dlg5 (w+; P{w[+mC]=UAS-CG6509.
GFP}3) under the control of the tubulin (tub)-Gal4 driver, active in
somatic cells of the ovary (Figure 1, B and B9). In escort and follicle
cells, the Dlg5::GFP protein is seen in puncta. In escort cells, Dlg5::
GFP accumulated on the extensions that protrude between the germline cysts in regions 2a and 2b (Figure 1, A and B9). In follicle cells, the
protein is mostly found on the apical side, facing the germ cells. Thus,
similar to vertebrates, polarized distribution of Dlg5 is conserved in
flies and hence may be functionally relevant.
Isolation of a dlg5 null allele by imprecise
P-element excision
To determine the effect of loss of Dlg5 on oogenesis, we set out to
generate a dlg5 null mutant. We mobilized the P{SUPor-P} insertion,
marked with yellow+ (y+) and white+ (w+) in dlg5 KG00748. This allele is
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homozygous lethal at late third instar larval stage, and also lethal in
late third instar larvae over Df(2L)BSC24, a chromosomal deletion that
uncovers dlg5. We obtained a total of 59 y w excision chromosomes,
of which 47 were viable over Df(2L)BSC242, indicating that the lethality observed on the original chromosome was due to the P-element
insertion. Twelve lines were confirmed to be lethal over Df(2L)
BSC242. Homozygotes and hemizygotes (over Df(2L)BSC242) of all
12 putative imprecise P-element excision mutant lines were embryonic lethal (see Materials and Methods). Although dlg5KG00748 mutants
are lethal during larval stages, dlg5D48 homozygous and hemizygous
animals die as embryos. Consequently, no larvae hatch, indicating
that dlg5 has an essential function in embryos, while dlg5KG00748
appears to represent a partial loss of function allele.
One of the lethal excision mutations, dlg5D48, was randomly selected
for further analysis. We collected single dlg5D48 embryos to map the
breakpoints of the deletion created by the P-element excision by PCR
(Supporting Information, Figure S1). Although we were able to detect
PCR fragments derived from DNA upstream of the P-element insertion
site in both wild-type and mutant embryos, we were unable to amplify
fragments downstream of it (primers 10 and 3; see Materials and Methods). Similarly, we were unable to amplify the region flanking the
P-element insertion site in dlg5D48 (primers 5 and 4). However, DNA
sequences distal to primer 3 could be amplified in both wild-type and
mutant DNA. The genes flanking dlg5, therefore, appear to be unaffected. We hypothesize that part of the 11-kb P-element may have undergone a rearrangement but that P-element-derived sequences remain
in the gene. We conclude that the sequences abutting the P-element
insertion site on the distal side are missing and that the distal deletion
breakpoint lies between primer 10 and primer 27. Together, these results
indicate that the N-terminal end of the protein is missing. Based on these
observations it is likely that dlg5D48 represents a null allele.
Dlg5 is required in the ovarian germline and soma
Because dlg5D48 is embryonic lethal, we used clonal analysis via FRTmediated site-specific mitotic recombination (see Materials and Methods) (Xu and Rubin 1993) to determine the requirement of dlg5 in the
germline and soma. Two types of clones, persistent and transient, are
observed in ovaries and are classified based on the time elapsed between clone induction and clonal analysis. Most clones observed 2 to
5 d ACI (after clone induction) are in the follicle cells of maturing egg
chambers. These transient clones originate from a recombination
event of a nonstem cell and are lost at the end of oogenesis when
the follicle cells disintegrate (Margolis and Spradling 1995). Persistent
clones result from a recombination event in a stem cell and will
typically last throughout the life of the fly as the stem cell continuously
self-renews with each division.
Clones were induced in newly eclosed females and analyzed 10 d
ACI (see Materials and Methods). In control flies, 48 ovarioles were
counted. Of these, 22 had persistent stem cell-derived germ line clones
and 17 had persistent clones derived from follicle stem cells (46% and
35%, respectively) (Figure 2C). Thirty-six ovarioles from ywP
[hs70FLP]; Ubi-GFP nls.2L FRT 40A/FRT 40A dlg5D48 flies were investigated for the presence of clones. No persistent dlg5 clones were
observed in the germline or follicle cells (Figure 2), indicating that dlg5
is essential for the normal division and possibly maintenance of both
germline and follicle stem cells. The percentage of ovarioles with
transient wild type and dlg5 clones were comparable for both time
points. After 5 d ACI, 64% of wild-type ovarioles (150 ovarioles)
contained transient clones, whereas dlg5 mutant clones were only
detected in 40% of ovarioles (150 ovarioles) (Figure 2C). Transient
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dlg5 clones were smaller than control clones (not shown), supporting
the idea that loss of dlg5 affects cell division or cell viability. From
these results we can conclude that Dlg5 is essential in both germline
and somatic stem cells, and it is likely required in all follicle cells
throughout oogenesis.
Reduced levels of Dlg5 in follicle cells leads to egg
chamber defects
Flies in which Dlg5 levels have been ubiquitously depleted by
expression of dlg5 RNAi under a tubulin (tub)-gal4 ubiquitous driver
die at the third instar larval stage with 2% of animals surviving into
the pupal stage. To circumvent the lethality associated with general
loss of Dlg5, RNAi expression was inhibited until after eclosion using
the Gal4-Gal80 system (Suster et al. 2004). Animals were reared at 18
and newly eclosed flies were held for 4 d at 29 to induce RNAi
expression before ovaries were dissected and immunostained for analysis. To address the possibility of off-target effects, the phenotype of
two different RNAi lines targeting dlg5 were compared (see Materials
and Methods). Both produced the same phenotypes, and so it is unlikely that the observed defects are a consequence of off-target effects.
Depletion of Dlg5 in the somatic cells of the ovary using the
general tub driver resulted in severe morphological defects. One striking phenotype was the overgrown stalk-like structures (Figure 1D,
arrowhead). Ovarioles containing degenerating follicles with only
nurse cell nuclei and surrounding follicle cells remaining were also
observed (Figure 1D, arrow).
Given the apical localization of Dlg5 in follicle cells, we depleted the
protein using the c587-Gal4 driver, which is most highly expressed in
somatic cells of the ovary, to drive expression of dlg5 RNAi (Figure 3A)
(Song et al. 2004). Females were transferred to 29 on eclosion to induce
RNAi expression and ovaries were dissected 4 and 10 d later. Experiments
were repeated at least twice and a minimum of 300 wild-type and KD
ovarioles were inspected. In 4-d-old females, 10% of ovarioles showed
normal morphology, whereas the other 90% contained abnormal germaria and egg chambers. The most striking phenotype was the overgrown
stalk-like structures observed in 84% of ovarioles (Figure 3B, arrow).
Some mutant egg chambers were separated by elongated stalks made
up of more than the usual four to six cells, whereas others were divided
by massively overgrown stalks that had failed to intercalate properly
(Figure 3B). In these abnormal stalks, instead of the typical single column
of cells, each stalk had at least two columns of cells. Although the organization of the stalks is aberrant, the cells themselves look similar to wildtype cells and have retained basal contact with their neighboring cell.
Fifteen percent of ovarioles contained at least one egg chamber
with two oocytes (Figure 3, C and C9). To confirm that the increased
nurse cell and oocyte number within enlarged egg chambers was not
the product of a defect in germline cell divisions, the number of ring
canals in each cell were counted. Each oocyte never contained more
than the expected four ring canals formed by four successive divisions
(Figure 3, C and C9), clearly indicating that defective packaging of
germline cysts by follicle cells underlies the "multiple cyst" phenotype.
The inclusion of multiple cysts within an egg chamber often results
from abnormal polar and stalk cell specification, as well as delayed
differentiation of these cell populations (Grammont and Irvine 2001;
Lopez-Schier and St. Johnston 2001). The observed stalk cell and polar
cell phenotypes suggest a defect at the level of cyst cell encapsulation.
In 10-d-old females, the ovaries had strikingly enhanced phenotypes
(Figure 3, D and E). At most 1% of ovarioles showed normal morphology and 50% of ovarioles contained either an abnormal-looking
germarium in an otherwise empty sac of somatic sheath cells or
Figure 2 Dlg5 requirement in germ
line and follicle stem cells. (A, A9) Control ovariole dissected 10 d after clone
induction (ACI), showing germline and
somatic persistent stem cell clones. (B,
B9) No germline or somatic dlg5D48
clones were detected in ovarioles
10 d ACI. (C) Graph noting the number
of ovarioles containing wild-type and
dlg5D48 persistent and transient clones
obtained by the original FRT technique (Xu and Rubin 1993). Clones in
(A, A9) are marked by the absence of
GFP and somatic cells by the presence
of Traffic Jam protein (red). DNA, blue.
a germarium with a few discrete egg chambers. In some cases, follicles
appear not to have separated from the germarium, and a number of
germline cysts appeared to be degenerating. Abnormal compound cysts
were found in the other 50%.
Together these results indicate the primary requirement of dlg5 in
follicle cells for the proper organization of the egg chamber as well as
stalk formation. The results suggest that abnormal patterning ultimately results in the loss of follicle cells. They further underline the
importance of the follicle epithelium to the integrity of the egg chamber and survival of germ line cells.
Loss of Dlg5 in follicle cells leads to ectopic polar cells
Fasciclin III (Fas III) is highly expressed in immature follicle cells until
egg chamber stages 2–3, when the level in most follicle cells is reduced
except in polar cells, which stand out due to their strong FasIII staining
(Figure 1A, B, and C). In addition to the observed stalk cell overgrowth,
many Dlg5 KD egg chambers displayed ectopic FasIII expression (Figure 1D). Closer examination showed clusters of FasIII-positive cells on
the surface of the follicular epithelium surrounding the egg chambers.
The location and characteristic shape of these cells suggested that they
were polar cell clusters. This was supported by further immunostaining, which revealed that these cells were negative for Eya (Figure 4B),
indicating that they had fully differentiated into polar cells. Eyes Absent
(Eya) is a repressor of polar cell fate and its absence is sufficient to
induce a polar cell fate (Figure 4) (Bai and Montell 2002; Chang et al.
2013). Typically, polar cells appear at the anterior and posterior termini
of each egg chamber (Figure 4A); however, in mutant egg chambers
large patches of up to 25 polar cells often appear at random on the
surface of the egg chamber (Figure 4B; Figure 3E, arrowhead). This
phenotype was observed in at least 5% of c587-Gal4-dlg5 RNAi ovarioles at d 4, and in .10% of egg chambers at 10 d.
Dlg5 is required for germ cell migration
Because of the requirement of dlg5 in migrating border cells in the
ovary (Aranjuez et al. 2012) and the embryonic lethal phenotype
associated with dlg5, we sought to investigate whether the gene is also
essential during germ cell migration and gonad morphogenesis in
embryos (for a detailed review see Kunwar et al. 2006). The primitive
embryonic gonad in Drosophila consists of the somatic gonadal precursor cells (SGPs) and the primordial germ cells (PGCs). These two
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Figure 3 Phenotypes resulting from depletion of Dlg5 in follicle cells. (A) Control ovarioles, driver only. (B) Dlg5 KD ovariole 4 d after RNAi
induction. Note the abnormal stalk between two Dlg5 KD egg chambers (arrows). (C) Dlg5 KD results in disorganized egg chambers with
abnormal numbers of oocytes. (C9) Enlarged picture of egg chamber with two oocytes. Both oocytes are connected by four ring canals with the
neighboring nurse cells, indicating the egg chambers contain two 16-cell cysts. (D) Ovarioles 10 d after RNAi induction. Most germaria and egg
chambers are disorganized and no mature egg chambers are detected. (E) Higher magnification of ovarioles 10 d after RNAi induction. The
packaging of the germline cells is abnormal. Note the patch of Eyes Absent (Eya)-negative follicle cells. (A, B, C, D, and E) Activity of the c587
driver (c587 . CD8::GFP) in the germarium is shown in green. (A, B) alpha-spectrin is shown in red, Eya is shown in green. (C9) Actin (phalloidin) is
shown in red. (C, C9) Orb (oocyte) is shown in green. (D and E) Eya protein shown in red. In all panels: DNA, blue; scale bar, 20 mm.
cell types are initially located in different regions of the embryo. The
SGPs are mesodermal in origin and are specified by zygotically active
genes during mid-embryogenesis in parasegments 10–13. By contrast,
the PGCs are formed by precocious cellularization at the posterior
pole of embryos at the syncytial blastoderm stage and are specified by
maternal determinants.
The PGCs follow a stereotypical trajectory to ultimately coalesce
with the SGPs into a gonad in a temporally coordinated manner. This
migratory journey begins at gastrulation, when the PGCs are carried
into the interior of the embryo by the mid-gut invagination. The
PGCs in each group migrate laterally to come into contact with the
gonadal mesoderm on either side of the embryo. PGCs align
themselves in a row with the SGPs in parasegments 10–13, and these
juxtaposed cells coalesce into the embryonic gonad. A combination of
repulsive and attractive cues guides PGC migration through the midgut and toward the SGPs (Van Doren et al. 1998; Santos and Lehmann
2004; Deshpande and Schedl 2005; Kunwar et al. 2006).
Because dlg5D48 embryos are lethal, we analyzed embryos derived
from dlg5D48/CyO en-LacZ flies to assess the requirement of dlg5 during
germ cell migration and gonad morphogenesis (Figure 5). Embryos were
stained with anti-Vasa and anti-B-galactosidase antibodies. Although
Vasa antibody labeled migrating germ cells, the B-galactosidase-specific
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staining allowed us to distinguish between the homozygous mutant
embryos from the balancer embryos, which served as controls (Figure 5).
In dlg5D48 embryos, PGC specification and earlier phases of germ cell
migration were relatively normal. Near wild-type numbers of PGCs were
internalized within the gut pocket and also successfully traversed through
the mid-gut epithelial wall. By stage 12 of embryogenesis, however, germ
cell migration defects became apparent, as in a subset of embryos, and
PGCs did not align with the SGPs. Consequently, several PGCs were
seen scattered in the posterior. Eventually, these PGCs were unable to
establish and/or maintain sustained contacts with the SGPs and were
detected several diameters away from the coalesced gonads. Fifty-six
percent of dlg5 embryos showed five or more scattered PGCs (n = 52)
as opposed to 17% of balancer embryos (n = 30). We also estimated the
average number of germ cells lost per embryo in an independent experiment wherein we focused on total number of surviving PGCs in control
as well as dlg5 embryos. The embryos between stages 13 and 16 were
used for the estimation. Consistently, we found that in the dlg5 embryos,
on average 4.4 PGCs per mutant embryo were lost (n = 18), as opposed
to 1.7 PGCs in the balancer embryos (n = 20).
Loss of PGCs or scattering during gonad coalescence could arise
due to multiple reasons, including misspecification or misalignment of
SGPs, defective transmission, and/or reception of guidance cues.
Figure 4 Polar cell numbers are increased in Dlg5 KD egg chambers. (A, A9) Control ovariole, driver only. Polar cells, marked by the absence of
Eyes Absent (Eya) staining (red), are highlighted. (B, B9) Dlg5 KD egg chamber. Note the increase of Eya-negative polar cells and the oocyte
(arrow) in the center of the egg chamber surrounded by four ring canals (actin, green). DNA, blue; scale bar, 20 mm.
Moreover, aberrant migration could result because of germ cell
autonomous defects or nonautonomous influence emanating from
the surrounding somatic tissue. To assess whether dlg-5 affects SGP
specification, we stained these embryos with antibodies against a SGP
marker, Eyes absent. Preliminary analysis suggests that total number
of Eya-positive cells in dlg5 embryos are comparable with that
detected in wild-type embryos, indicating that SGP specification is
likely not affected in dlg5 embryos. Although this observation points
to a specific requirement of dlg5 during gonad coalescence, detailed
analysis will be necessary to address the precise cause underlying loss
of PGCs and whether dlg5 function is needed in the somatic component of the gonad, i.e., SGPs or PGCs or in both cell types.
Loss of Dlg5 leads to defects in E-cadherin distribution
To determine if the polarization of follicle cells is generally disrupted,
we investigated the distribution of three proteins that are localized to
specific membrane domains. The distribution of FasIII in the lateral
membrane of Dlg5 KD cells was indistinguishable from wild-type
(Figure 6, A, A9, B, and B9) (Nystul and Spradling 2010). The concentration of Bazooka (Baz) on the apical side of follicle cells was also
not affected in Dlg5 KD cells (Figure 6, A, A9, A99, B, B9, and B99)
(Abdelilah-Seyfried et al. 2003). It was therefore noteworthy that the
distribution of DE-cadherin, the Drosophila homolog of E-cadherin,
was less organized in Dlg5 KD follicle cells than in wild-type, giving
an impression of lower levels of protein. Further, although E-cad
showed high levels of protein in wild-type and mutant polar cells,
the asymmetric accumulation of the protein was disrupted in Dlg5
KD polar cells (Figure 6 C, C9, C99, D, D9, D99).
The disruption of E-cadherin accumulation may contribute to the
disorganized appearance of Dlg5 depleted ovaries. E-cadherin is
typically upregulated in posterior follicle cells, and the oocyte
preferentially associates with follicle cells expressing higher levels of
DE-cadherin as the developing cyst enters region 3 (Godt and Tepass
1998; Gonzalez-Reyes and St. Johnston 1998). Oocyte mislocalization
is frequently observed in females that have depleted Dlg5 levels in
follicle cells (Figure 3C, C9). In some cases, the oocyte fails to associate
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949
Figure 5 Dlg5 embryos show defects
in gonad formation. Dlg5D48 embryos
show variable germ cell migration
defects and germ cell loss. While properly coalesced gonads are visible in
the mutant embryos, several germ
cells are scattered away from the coalesced gonads. Dlg5D48/CyO en-lacZ,
control embryos recognized by the
striped beta galactosidase staining. All
embryos are approximately stage 13.
at all with surrounding follicle cells and instead is located in the center
of the nurse cells (Figure 4B9, arrow). Instances have also been observed in which the oocyte is completely mislocalized within the
follicle (i.e., a single oocyte is found at the anterior termini of the
follicle), yet remains associated with follicle cells (not shown).
DISCUSSION
We found that dlg5 is essential for normal division or maintenance of
FSCs and GSCs, and is required in later stage follicle cells. Reduction
of Dlg5 levels disrupts egg chamber formation, indicating that dlg5 is
involved in processes fundamental to egg chamber organization.
When Dlg5 was depleted in follicle cells for 4 d, epithelial follicle
cells showed relatively normal cell shape and polarity, but the polar
and stalk cells showed strong abnormalities in number, localization,
and overall organization. The induction of ectopic polar cells and stalk
cell overgrowth observed in these ovaries is significant because it has
been suggested that the differentiation of these two cell types are
controlled by similar signaling events, which distinguishes them from
the other follicle cells (Tworoger et al. 1999; Zhang and Kalderon
2000). Loss of Dlg5 may interfere with the pathways that specify stalk
and polar cell differentiation and maturation and, consequently, egg
chamber organization.
The significantly enhanced phenotype observed upon depletion of
Dlg5 in follicle cells for 10 d agrees with the dlg5D48 clonal phenotype
and is consistent with the proposition that the function of dlg5 is
completely lost in dlg5D48. Further, complete loss of dlg5 function
results in embryonic lethality, possibly also as a result of abnormal
organization and integration of specific embryonic cells.
Our analysis of primitive embryonic gonad formation in dlg5D48
embryos suggested that it likely functions during germ cell migration
and gonad coalescence. These data are reminiscent of requirements of
Dlg5 in the maintenance of the cohesion of migrating border cells in
the ovary (Aranjuez et al. 2012). In this regard, it is interesting to note
that proper gonad coalescence has been shown to depend on cell
adhesion molecule E-cadherin (Jenkins et al. 2013). E-cadherin is
consistently upregulated during late stages of gonad formation. Because loss of dlg5 results in altered distribution of E-cadherin in follicle
cells, it is conceivable that aberrant E-cadherin levels and/or localization could also contribute to the embryonic gonad-specific phenotypes. In this context, it will be interesting to analyze further the
precise requirement and cell-type specificity of Dlg5 function in controlling cell adhesion and migration.
Many of the dlg5 phenotypes we have described here could result from
aberrant E-cadherin distribution. For instance, oocyte mislocalization
Figure 6 E-cadherin, but not Fasciclin III or Bazooka, is mislocalized in Dlg5 KD follicle cells. (A, A9) Control ovariole, driver only, and (B, B9) Dlg5 KD
ovariole both show normal distribution of FasIII and Bazooka (higher magnification in A99 and B99). (C, C9) E-cad is more tightly localized in control
ovarioles compared to its distribution in Dlg5 KD ovarioles. E-cad is asymmetrically localized on the apical side in control polar cells, but it looks
uniformly distributed in Dlg5 KD polar cells (C9, C99 and D9, D99 show higher-resolution images of two examples). DNA, blue, scale bar, 20 mm.
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E. Reilly et al.
is often seen as a result of loss or aberrant homophilic adhesion mediated by E-cadherin between posterior follicle cells and the oocyte (Godt
and Tepass 1998; Gonzalez-Reyes and St. Johnston 1998). Formation of
interfollicular stalks is also dependent on dynamic E-cadherin accumulation: E-cadherin accumulates first at the apical boundary of prefollicular cells, followed by the establishment of lateral cell contacts to initiate
and complete intercalation to form a single wide stalk (Besse et al.
2002). Additionally, E-cadherin has been demonstrated to be required
for recruiting and anchoring stem cells to their niche prior to adulthood
in the Drosophila ovary (Song and Xie 2002; Song et al. 2002). By clonal
analysis, we showed that both germline and follicle dlg5 ovary stem cells
are unable to give rise to normal daughter cells, indicating that the gene
is essential in these cells. This may suggest several possibilities. There
may be a cell-autonomous requirement for dlg5 in follicle cells; however,
the loss of stem cells may also be an indication of the requirement for Ecadherin-mediated adhesion to the stem cell niche. Finally, the lack of
cohesion in migrating germ cells in dlg5D48 embryos is consistent with
a perturbation in cell–cell adhesion, as described above. The observed
phenotypes, therefore, and the role of Dlg5 in E-cadherin distribution
may be related; however, more work is needed to determine the nature
of the participation of Dlg5 in E-cadherin distribution before any further conclusions may be drawn.
Dlg5 is involved in vesicle trafficking in vertebrates and has been
reported to colocalize with several Rab GTPases (Nechiporuk et al.
2007; Chang et al. 2010). The punctate distribution of Dlg5 in the
Drosophila ovary is consistent with a similar association of the protein
with endosomes. Therefore, it seems possible that Dlg5 is involved in
endosomal trafficking. The abnormal distribution of E-cadherin,
which is recycled through the endosome, observed in Dlg5 KD ovaries
supports this hypothesis (Jafar-Nejad et al. 2005; Langevin et al. 2005).
Further, reduction of Dlg5 in the mammalian epithelial cell line LLcPK1 resulted in lower levels of E-cadherin, but it is not clear how Dlg5
controls E-cadherin levels (Sezaki et al. 2012).
Thus, Dlg5, like its human homolog and Dlg1, may be involved in
endosomal recycling of E-cad. But based on this characterization of
dlg5 and its functional requirement, there are fundamental differences
between these two genes. Although dlg5 shows embryonic lethality
and is essential in ovarian stem cells, dlg1 larvae can survive for .10
d and show overgrowth phenotypes in larvae and follicle cells. Persistent dlg1 germ line clones develop into eggs, whereas follicle cells
lacking dlg1 sometimes show tumor-like invasion into the interior
of the egg chamber (Goode and Perrimon 1997), a phenotype we
did not observe in Dlg5 KD. The task of figuring out what each of
these Dlg proteins contributes to apical protein trafficking and cell
survival should prove informative and stimulating.
ACKNOWLEDGMENTS
We thank D. Godt, S. DiNardo, and T. Schüpbach for fly stocks and
antibodies. We are grateful to S. Minakhina, S. Radford, and C. Rauskolb
for helpful discussion of the manuscript, and to L. Nguyen for fly food.
This work was supported by grants from the National Institutes of
Health (RO1GM089992 to R.S. and RO1GM110015 to G.D.) and by
the W. Horace Goldsmith Foundation.
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Communicating editor: H. D. Lipshitz